Ismail Ustunel

The effect of testosterone on gastrocnemius muscle fibres in growing and adult male and female rats: a histochemical, morphometric and ultrastructural study.

In this study, the effect of testosterone on gastrocnemius muscle fibres in growing and adult rats (male and female) was examined using histochemical, morphometric and ultrastructural techniques.

Uterine FK506-binding protein 52 (FKBP52)–peroxiredoxin-6 (PRDX6) signaling protects pregnancy from overt oxidative stress

Immunophilin FK506-binding protein 52 (FKBP52) is a cochaperone that binds to the progesterone receptor (PR) to optimize progesterone (P4)-PR signaling. We recently showed that Fkbp52-deficient (Fkbp52−/−) mice have reduced uterine PR responsiveness and implantation failure which is rescued by excess P4 supplementation in a genetic background-dependent manner. This finding led us to hypothesize that FKBP52 has functions in addition to optimizing PR activity. Using proteomics analysis, we found that uterine levels of peroxiredoxin-6 (PRDX6), a unique antioxidant, are significantly lower in Fkbp52−/− mice than in WT and PR-null (Pgr−/−) mice. We also found that Fkbp52−/− mice with reduced uterine PRDX6 levels are susceptible to paraquat-induced oxidative stress (OS), leading to implantation failure even with P4 supplementation. The same dose of paraquat did not interfere with implantation in WT mice. Moreover, treatment with antioxidants α-tocopherol and N-acetylcysteine (NAC) attenuated paraquat-induced implantation failure in P4-treated Fkbp52−/− mice. Functional analyses using mouse embryonic fibroblasts show that Fkbp52 deficiency associated with reduced PRDX6 levels promotes H2O2-induced cell death, which is reversed by the addition of NAC or by forced expression of PRDX6, suggesting that Fkbp52 deficiency diminishes the threshold against OS by reducing PRDX6 levels. These findings provide evidence that heightened uterine OS in Fkbp52−/− females with reduced PRDX6 levels induces implantation failure even in the presence of excess P4. This study shows that FKBP52–PRDX6 signaling protects pregnancy from overt OS.

The immunohistochemical localization of notch receptors and ligands in human articular cartilage, chondroprogenitor culture and ultrastructural characteristics of these progenitor cells

The presence of progenitor/stem cells in human articular cartilage remains controversial. Therefore, we attempted to isolate and culture progenitor/stem cells and to investigate their phenotypic characteristics. Biopsies were obtained (with consent) from patients undergoing arthroscopic surgery. Full depth explants were fixed and cryosectioned or enzymatically digested and the resulting cells cultured and plated on fibronectin-coated dishes. Chondrocytes were cultured until colonies of >32 cells were present. Colonies were trypsinized and expanded in monolayer for pellet culture. Immunolocalization of Notch and its ligands were detected in vivo and in vitro using immunocytochemistry. In vitro studies investigated differences in immunolocalization of Notch and its associated ligands in colony-forming cells and small clusters of non-colony-forming cells. The ultrastructure of the chondroprogenitors was examined by scanning and transmission electron microscopy. Results revealed that the immunolocalization of Notch-1 and its ligand Delta were concentrated in regions closest to the articular surface. Notch-1 was also densely localized in the deeper zone of articular cartilage. Notch-2 immunolabeling was densely localized in all zones of articular cartilage. Jagged-1 was concentrated in the deeper regions of articular cartilage. Notch-1, Delta and Jagged-1 were more abundant in colony-forming cells than non-colony-forming chondrocytes in vitro. Notch-3, Notch-4 and Jagged-2 were absent from all regions of the articular cartilage tissues and cultured cartilage cells in vitro. Ultrastructurally, chondrocytes cultured in monolayer dedifferentiated to fibroblast-like cells with cell surface processes of varying lengths, pellet cultured cells varied in morphology, as flattened and rounded. In conclusion, we propose that adult human articular cartilage may contain cells having progenitor cell features.

Distribution of Notch family proteins in intrauterine growth restriction and hypertension complicated human term placentas.

Members of the Notch family have been detected in many developmental and cell specification processes during placental development. However, Notch protein expression in Intrauterine Growth Restriction (IUGR) and Pregnancy Induced Hypertension (PIH) is not clear. In this study we aimed to clarify the immunolocalization of Notch proteins in full-term placentas after IUGR and PIH in comparison with normal placentas. Formalin-fixed, paraffin-embedded term placentas obtained by caesarean operations were processed for immunohistochemical localization of Notch 1, 2, 4 and Jagged 2. Transmission electron microscopy was also performed. In normal term placentas, all Notch proteins were intensely immunostained in the brush border of cells of the syncytiotrophoblast layer of the basal (maternal) side and the chorionic plate (fetal) side. The endothelial cells were also intensely immunostained in both sides for Notch 1. However, in IUGR and PIH placentas, the immunoreactivities of all Notch proteins were decreased significantly in the brush border of cells of the syncytiotrophoblast layer and the reaction was generally observed in the cytoplasm of syncytiotrophoblast cells in the basal and chorionic plate sides. The reactivity in endothelial cells was also significantly decreased. Our results have shown that the immunoreactivity and localization of Notch proteins is altered in pathologic placentas. Therefore, we propose that deregulated expression of Notch proteins may contribute to the disruption of trophoblast differentiation, endothelial cell function and/or feto-maternal traffic down-regulation during pregnancy or vice versa in such pathologic conditions.

Recurrence of keratoconus in two corneal grafts after penetrating keratoplasty.

Purpose: To report the recurrence of postkeratoplasty keratoconus in 2 corneal grafts harvested from the same donor. Design: Interventional case reports. Methods: A 21-year-old-man with advanced keratoconus in his right eye and a 28-year-old-woman with corneal leucoma in her right eye underwent penetrating keratoplasty with 2 grafts coming from the same donor. Approximately 1.5 years after grafting, corneal irregularity and astigmatism caused visual acuities of the patients to decrease to counting fingers. Clinical findings and corneal topography suggested the recurrence of keratoconus. A repeat keratoplasty was performed in both patients. Results: Histopathology of the excised corneal grafts was consistent with keratoconus and confirmed the preoperative diagnosis. Conclusions: Recurrence of keratoconus in a patient who had no preexisting keratoconus and in 2 corneal grafts coming from the same donor suggested transmission of the disorder from the donor instead of true recurrence.

Melatonin Is as Effective as Testosterone in the Prevention of Soleus Muscle Atrophy Induced by Castration in Rats

The purpose of this experiment was to compare the weight, insulin‐like growth factor‐I (IGF‐I) expression, and ultrastructure of the soleus muscle in growing castrated rats treated with testosterone or melatonin. In this study, adult male Wistar albino rats were used. The groups were arranged as sham, castrated, and testosterone‐ or melatonin‐injected groups after castration. The soleus muscle samples were fixed in Bouins solution for immunohistochemistry, and in 2.5% gluteraldehyde in 0.1 M phosphate buffer (pH 7.4). Whereas castration reduced the soleus weight and fiber diameter, testosterone and melatonin administration increased them. IGF‐I immunostaining observed in the satellite cells and periphery of the myofibers was least intense in the castrated group. Strong staining of IGF‐I was observed in the testosterone‐ and melatonin‐administered groups. The ultrastructure of the soleus muscle in castrated animals showed the important ultrastructural modifications related to degeneration. In these groups, degenerative mitochondria, glycogen clusters under the sarcolemma, irregular Z lines, and loss of lamina externa were observed. The ultrastructure of myofibrils in the testosterone‐ and melatonin‐injected groups was similar to that in sham groups in view of structure. In conclusion, we suggest that melatonin is as effective as testosterone in the prevention of atrophy induced by castration through the IGF‐I axis. Anat Rec, 291:448–455, 2008.

Endogenous orexin-A modulates gastric motility by peripheral mechanisms in rats

Orexin-A (OXA) and orexin receptor type 1 (OX1R) are found in enteric nervous system and smooth muscle cells in the digestive tract. Fasting is a stimulant for OXA synthesis. The aim of the present study was to investigate central and peripheral effects of endogenous OXA on gastric motility. Endogenous OXA synthesis was induced by 36h fasting. Vagotomy was used to evaluate N.vagus-mediated effects of OXA. Gastric emptying and interdigestive gastric motility were measured by spectrophotometric and manometric methods, respectively. Rats were pretreated with OX1R antagonist SB-334867 prior to measurements. Plasma OXA concentration was assayed with radioimmunoassay while preproorexin (PPO) expression was determined with Western blotting in gastric and hypothalamic tissues. OXA immunoreactivity in antrum was determined with immunohistochemistry. Plasma OXA level, PPO protein expression and OXA immunoreactivity were significantly increased in response to 36h fasting. Endogenous OXA facilitated gastric emptying and inhibited gastric interdigestive motility. As these effects were abolished with SB-334867, it is likely that gastrokinetic effects of OXA are mediated via OX1R. Vagotomy did not alter OXA-mediated effects. According to current data, OXA is up-regulated both centrally and peripherally upon fasting. Endogenous OXA accelerates gastric emptying while it inhibits interdigestive motility.

Is there a relationship between PCNA expression and diabetic placental development during pregnancy

We aimed to investigate the distribution pattern of proliferating cell nuclear antigen (PCNA) by immunohistochemistry and Western blot in placentas of control and diabetic rats at different stages of pregnancy. It is still not clear how proliferation is coordinated and how this coordination is affected by diabetes in the placenta. Diabetes was induced by streptozocin on the first day of pregnancy. Animals were sacrificed on days 11, 13, 17 and 21 of pregnancy. In control placentas immunolabeling intensity of PCNA was the highest on days 11 and 13 of pregnancy and decreased with progression of pregnancy. In the diabetic groups immunolabeling was less intense on days 11 and 13 of pregnancy compared to controls. However, in parallel with placental weights, PCNA immunopositivity was more intense in diabetic groups than control groups on days 17 and 21 of pregnancy, and the difference was statistically significant on day 17. According to Western blot data, on days 11 and 13 of pregnancy the amount of PCNA was greater in control groups than in the diabetics, whereas it was greater in diabetic groups than the controls on days 17 and 21 of pregnancy. We conclude that PCNA may play a role in abnormal placenta formation resulting from diabetes.

Effects of formaldehyde inhalation on the junctional proteins of nasal respiratory mucosa of rats

Exposure to formaldehyde, which is an organic compound, disturbs the integrity of nasal mucosa. In this study, we aimed to clarify the protein changes in the junctional complex of nasal mucosa of Wistar rats exposed to formaldehyde inhalation. The study was performed in 20 female Wistar rats. Rats were divided into two groups randomly. Control rats were allowed free access to standard rat chaw and tap water (n:10). Experimental group was exposed to formaldehyde vapor at 15ppm, 6h/day, 5 days/week for 12 weeks (n:10). Histological evaluation of the experimental model was determined by hematoxylin-eosin (HE) and periodic acid Schiff (PAS) stainings of paraffin-embedded nasal mucosa tissues and by electron microscopy. The effects of formaldehyde inhalation on the distribution of occludin, E-cadherin, and gamma-catenin were assessed by immunohistochemistry. The nasal mucosa of the experimental group was correlated with hypertrophy in goblet cell, degeneration in basal lamina, stratification of epithelium, and proliferation. Thickness of basal lamina and also local degenerative regions, vacuole increase in cytoplasmic areas, irregular forms of kinocilium and loss of sharpness in the kinocilium membrane were the findings at the ultrastructural level. The expressions of E-cadherin, occludin, gamma-catenin proteins in intercellular junctional complexes of rat nasal mucosa were also decreased in experimental group compared to control group. The findings of the present study indicated that formaldehyde vapor inhalation in the concentrations and duration of exposure used in the present experiment significantly decreased the density of structural proteins of the junctional complex in the nasoepithelium. It was suggested that, the formaldehyde inhalation could cause complete impairment of intercellular junctional complexes and disturb the tissue integrity in nasal mucosa at higher concentrations.

Dynamic Hedgehog signalling pathway activity in germline stem cells

Although the contribution of Hedgehog (Hh) signalling to stem cell development and oncogenesis is well recognised, its importance for spermatogonial stem cells (SSCs) has not been established. Here we interrogate adult rat SSCs using an established model in which only undifferentiated spermatogonial cells remain in the testis at 15 weeks following irradiation, and spermatogonial differentiation is induced within 4 weeks by gonadotrophin‐releasing hormone antagonist (GnRH‐ant) administration. Synthesis of Hh pathway components in untreated adult rat testes was compared with that in irradiated testes prior to and after GnRH‐ant exposure using in situ hybridization. In adult testes with complete spermatogenesis, the Desert Hedgehog ligand transcript, Dhh, was detected in Sertoli cells, some spermatogonia and in spermatocytes by in situ hybridization. Spermatogenic cells were identified as sites of Hh signalling through detection of transcripts encoding the Hh receptor, Ptc2 transcripts and proteins for the key downstream target of Hh signalling, Gli1 and the Hh transcriptional activator, Gli2. Remarkably, the undifferentiated spermatogonia present in irradiated adult rat testes contained Dhh in addition to Ptc2, Gli1 and Gli2, revealing the potential for an autocrine Hh signalling loop to sustain undifferentiated spermatogonial cells. These transcripts became undetectable by in situ hybridization following GnRH‐ant induction of spermatogonial differentiation, however, detection of Gli1 protein in spermatogonia in all groups indicates that Hh signalling is sustained. This is the first evidence of active Hh signalling in mammalian male germline stem cells, as has been documented for some cancer stem cells.